1. Field of the Invention
The process that is presented relates to confocal and 2-photon fluorescence microscopy as described by M. Gxc3x6ppert-Mayer, Ann. Physik 9, 273 (1931) and T. Wilson, Theory and Practice of Scanning Optical Microscopy (Academic Press, 1984). Both methods are assumed to be known.
Both confocal fluorescence microscopy and 2-photon microscopy are modified by the method described below to the extent that an additional contrast parameter is possible: the fluorescence lifetime.
2. Related Art
Temporal resolved fluorescence and/or the use of the lifetime as a contrast parameter in confocal/2-photon microscopy can be carried out by two different methodsxe2x80x94by time domain detection and frequency domain detection.
In the case of time domain detection (described in Published patent application DE 41 04 014 of Wabnitz, entitled xe2x80x9cMethod for Determining the Calcium Concentration in Cellsxe2x80x9d; and in Patent DE 36 14 359 C2 of Grxc3x6bler, entitled xe2x80x9cDevice for the Analysis and Imaging of Real Time Intensity of Fluorescence Radiation resulting from Point-by-Point Excitation of a Preparation by means of Laser Lightxe2x80x9d) a fluorescent sample is excited so as to produce fluorescence by means of a pulsed light source, and the fluorescence emission is detected with time resolution either by means of time correlated single photon counting (TCSPC)(described in Patent WO 98/09154 to Mxc3xcller et al., entitled xe2x80x9cSystem for Differentiating Fluorescing Molecular Groups by means of Time-Resolved Fluorescence Measurementxe2x80x9d; and in Published patent application DE 42 31 477 A1 of Han, entitled xe2x80x9cMethod for Optical Sorting of Plastics by means of Time-Resolved Laser Spectroscopyxe2x80x9d) or by means of time gated detection (described by H. Schneckenburger et al., xe2x80x9cTime-Gated H. Microscopic Imaging and Spectroscopy in Medical Diagnosis and Photobiology,xe2x80x9d Optical Engineering 33 (8) 2600 (1994); R. Cubeddu et al., xe2x80x9cA Real Time System for Fluorescence Lifetime Imaging,xe2x80x9d SPIE 2976 (1997) 98; and K. Dowling et al., xe2x80x9cTwo Dimensional Fluorescence Lifetime Imaging using a 5 kHz/110 ps Gated Image Intensifierxe2x80x9d (www.kentech.co.uk/pdf_files/K_Dowling_et_al.pdf)).
In the case of frequency domain detection, a fluorescent sample or preparation is excited with a light source that is either actively modulated or pulsed (for example, by means of passive mode coupling). Since any arbitrary modulation of the excitation by means of a Fourier analysis breaks down into sinusoidal components, the observation of a sinusoidal excitation is adequate. The frequency domain detection technique is based on the delay of the fluorescence emission by a phase f and a change in the modulation depth M compared with the excitation light as a function of the modulation frequency xcfx89(=2 xcfx80fmod) and the lifetime xcfx84.
xcfx86=a tan(xcfx89xcfx84)xe2x80x83xe2x80x83(1)
                    M        =                  1                                    1              +                                                ω                  2                                ⁢                                  τ                  2                                                                                        (        2        )            
However, the resulting fluorescence signal, oscillated with the modulation frequency, is out of phase and demodulated. For typical fluorescence lifetimes ranging from xcfx84=1 . . . 10 ns, modulation frequencies ranging from fmod=10 . . . 100 MHZ are adequate.
Since it generally does not make any sense to scan the fluorescence signal at such high modulation frequencies, a frequency mixing process is used to detect the signal. For mixing, any detection element with a modulatable amplification is suitable.
In essence two methods are distinguishedxe2x80x94the homodyne and the heterodyne detection techniques.
To understand the principle, one observes generally two xe2x80x9csignalsxe2x80x9d S1, S2, (where, for example S1 is modulated excitation, and S2 is modulated amplification).
S1=A0+A1 cos(xcfx89at+xcex1)
S2=B0+B1 cos(xcfx89bt+xcex2)xe2x80x83xe2x80x83(3)
Multiplication results in:
S1S2=A0B0+A0B1 cos(wbt+b)+B0A1 cos(wat+a)+A1B1{cos((wawb)t+(a+b))+cos((wawb)t+(axe2x88x92b))}xe2x80x83xe2x80x83(4)
If xcfx89a=xcfx89b (homodyne), the second harmonic and a frequency independent component are generated by the mixing process of the xe2x80x9csignalsxe2x80x9d S1 and S2. A low pass filter results in a suppression of the components at xcfx89a and 2xcfx89a. Only the DC background and the phase dependent DC component are detected. The signal, filtered by means of a low pass (LP) filter, can be written as:
xe2x80x83LP(S1S2)=A0B0+A1B1cos(xcex1xe2x88x92xcex2)xe2x80x83xe2x80x83(5)
In the case of homodyne detection this frequency independent (DC) signal can be detected in multiple relative phases. To measure the fluorescence lifetime, at least 3 three different phase positions are necessary. At just two relative phase positions, the phase shift or the demodulation, induced by the fluorescence lifetime, can be used as the contrast parameter (as described by P. C. Schneider et al., xe2x80x9cRapid Acquisition, Analysis and Display of Fluorescence Lifetime Resolved Images for Real time Applications, xe2x80x9d Rev. Sci. Instrum. 68 (11) 4107 (1997) (hereafter, xe2x80x9cSchneider et al.xe2x80x9d)).
If xcfx89b=xcfx89a+Dw (heterodyne), the mixing process generates a high frequency signal at the total frequency and a signal at the cross correlation frequency xcex94xcfx89. Again the high frequency components are suppressed by a low pass filter.
LP(S1S2)=A0B0+A1B1 cos(xcex94xcfx89t+xcex1xe2x88x92xcex2)xe2x80x83xe2x80x83(6)
In the case of heterodyne detection, the differential frequency xcex94xcfx89 is detected. Phase position and modulation depth of the signal at the differential frequency make it possible to determine the lifetime. Typical cross correlation frequencies range a few Hz up to about 100 kHz.
A more comprehensive presentation of the heterodyne method can be found in the publication by E. Gratton et al., xe2x80x9cA Continuously Variable Frequency Cross Correlation Phase Fluorometer with Picosecond Resolution,xe2x80x9d Biophys. J. 44 (1983) 315 (hereinafter, xe2x80x9cGrattonxe2x80x9d), and xe2x80x9cMultifrequency Phase and Modulation Fluorometer, xe2x80x9d Ann. Rev. Biophys. Bioeng. 13 (1984) 105 (hereinafter, xe2x80x9cGratton 2xe2x80x9d).
In both the homodyne and heterodyne detection technique the high frequency change is reflected, so to speak, on the low frequency range.
Owing to the multi-exponential decay behavior of the fluorescence emission, the lifetimes, determined from the modulation depth and the phase shift, vary. Therefore, for precise measurement of the lifetime, the excitation frequency has to be varied as described by Gratton 2.
If, in contrast, the lifetime is supposed to be used, for example, as a contrast parameter in an imaging process, this is generally not necessary. Frequently it suffices, for example, to show the phase shift or the demodulation by means of the lifetime or by means of the lifetime, calculated from the phase shift and demodulation data, at a fixed modulation frequency.